The immediate objective of the proposed research program is to relate nucleotide sequence to function in several regions of the simian virus 40 genome believed to contain regulatory functions. The first region to be studied will be the SV40 origin of DNA replication. This study will utilize previously constructed, viable variants of SV40 which contain two functional origins of DNA replication. Deletion and point mutations will be introduced in the region containing the origin of DNA replication, the mutants will be assayed for altered function, and the alteration in the nucleotide sequence will be determined. Similar studies will be carried out in regions believed to contain promotors for viral transcription, regions surrounding know "splice" points in "early" SV40 mRNAs, and the region which encodes the 3' noncoding segments of both "early" and "late" SV40 mRNAs.